Dicitrate cyclic diester dentifrice

ABSTRACT

Dicitrate cyclic diester (dicitrate) is a novel compound which is found in patients who do not exhibit symptoms or predisposition to idiopathic renal calculous disease. Dicitrate can be isolated and detected by novel chromatographic methods. Further, treating patients prophylactically and therapeutically with dicitrate cyclic diester can prevent or reduce the effects of calcification-related disease, including some types of kidney stones, or other disease or condition associated with the presence of metal ions. The metal ion complexing properties exhibited by dicitrate are also applicable to the use of the subject compounds as chelating agents and methods of binding, and thereby reducing, the amounts of metal ions in a substance.

This invention was made with government support under NationalInstitutes of Health Research Grant No. P01-DK20586; and with supportfrom Veteran Affairs Medical Center, Gainesville, Fla. The governmenthas certain rights in this invention.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a division of application Ser. No. 08/261,581 filedJun. 6, 1994 and is a continuation-in-part of application Ser. No.08/080,017, filed Jun. 18, 1994, now U.S. Pat. No. 5,376,553.

BACKGROUND OF THE INVENTION

Renal calculous disease is a common disorder in this country which hasbeen suggested to be on the rise. In 1950, only 0.95 per one thousandAmericans were estimated as having the disease. By 1984, this number hadrisen to 1.64 per one thousand Americans. Boyce, W. H. [1979]"Epidemiology of Lithiasis in the United States," XVIIIth C. M. Congressof the International Society of Urology, Paris, Kongressbericht, Tome 1,pp. 79-86. A 1978 report by Sierakowski et al. estimated that about 12%of the U.S. populace will suffer from stone disease at least once intheir lifetime (Sierakowski, R., B. Finlayson, R. R. Landes, C. D.Finlayson, N. Sierakowski [1978] Invest. Urol. 16:438-441). Thatestimate would be expected to have increased substantially in accordancewith the increased incidence of kidney stone disease.

The most prevalent type of renal stone disease is of a calcareous andidiopathic nature, occurring mostly in white males. Therefore, it isknown that incidence of kidney stone disease is associated with genderand race differences. Geographic and dietary factors can also affectincidence.

Unlike normal biomineralization processes (e.g., bone and toothformation), calcium oxalate urolithiasis is a pathological process.Although this process has been extensively studied, adequate therapy forthe disease can be a highly complex and uncertain subject to the averagemedical practitioner. See Thomas, Jr., W. C., [1976] Renal Calculi: AGuide to Management, Charles C. Thomas, Publisher, Springfield, Ill.,177 pp. In addition, kidney stone disease can be an extremely painfulexperience for the patient. Passage of stones can cause renal colic,destruction of kidney tissue from mechanical irritation by the stone, aswell as back pressure from obstruction of urinary flow. An increasedrisk of infection also accompanies the presence of any foreign body.

Crystallization within the urinary tract occurs opportunistically andquite freely. Formation of smaller crystallites is a normal renalfunction for eliminating calcareous stone salts. Abnormal conditionspresumably result from uncontrolled crystal agglomeration and/orcellular attachment/retention of crystals. Stone disease arises whencrystallite attachment is not blocked within the urinary tract.Consequently, further biomineralization and accretion of other celldebris and solutes create flow-obstructing kidney stones. Althoughcertain factors, e.g., uncontrolled biosynthesis of oxalate(hyperoxaluria), elevated phosphate levels (phosphaturia), and excessivedietary intake of oxalate-rich foods can exacerbate stone formation, itremains an idiopathic disease.

The prevalence of kidney stone disease arising from pathological calciumoxalate biomineralization contributes substantially to the cost ofhealth care in the United States. For example, costs for hospitalizationand surgery or extensive outpatient extracorporeal shock-wavelithotripsy treatment of approximately 500,000 U.S. residents annuallyhas been estimated at $3 billion. These figures exclude lostoccupational time during treatment or recuperation. Davis et al. (Davis,G. K., N. B. Cummings, B. Finlayson, J. L. Meyer, M. J. V. Smith [1974]"Urolithiasis," in Geochemistry and the Environment, Natl. Acad. Sci.,Washington, pp. 133-138) put the number of lost working days at nearly15,000,000 annually in this country alone, which translates to around56,000 person-years of productivity lost each year. An even more tellingexpression of the economic impact is that a work force of 56,000corresponds to the productivity of an entire city of about 130,000 to150,000. Thus, it is clear that a simple, accurate, and reproduciblemethod for detecting or treating such conditions, even in advance ofstone formation, is a valuable contribution to the management of kidneystone disease.

Previously, methods for detecting kidney stone formation or treatingkidney stone disease have been described, but are quite different fromthe subject invention. For example, U.S. Pat. No. 5,137,722 describes anextract and pharmaceutical composition for treatment of calcium oxalatestone disease. The extract is purified from the plant Edobotryajaponica. The extract does not consist of any citrate-containingcompounds.

U.S. Pat. No. 4,399,003 describes a method and kit for diagnosing apatient's proneness to develop calcium oxalate-type kidney stones. Themethod comprises measuring the rate of decrease of calcium ionconcentration in the patient's urine sample as compared to a referencestandard of normal urine. This method differs substantially from thediagnostic method described hereinbelow, which does not directly measurecalcium ion concentration in the patients' urine.

U.S. Pat. No. 4,888,182 also describes methods and compositions for thetreatment and prophylaxis of calcium renal stones. By contrast, thesubject invention comprises a novel compound and methods of use whichare clearly distinguishable from the titrate salt and methods of usingthat ionic salt as described in the '182 patent.

Except for a small fraction of stone-formers (e.g., individuals withprimary hyperoxaluria, those with renal tubular acidosis, and othershaving chronic hypercalcemic disorders), there has been no clearlydefined single criterion, or uniform set of criteria, that can becharacterized for the much larger group of idiopathic kidneystone-formers, which are addressed by the subject invention.

A few other potential diagnostic tools for discerning urolithiasis havebeen described in the scientific or medical literature. Illustrative arethe work of Zhmurov (Zhmurov, V. A. [1991] Urol. Nefrol-Moscow May-June(3):12-15), who found altered phospholipid composition in the urine ofstone-formers, and Azoury et al. ([1990] Urolog. Res. 18:7-11) whodescribed the use of nuclear magnetic resonance proton-relaxation-rates(PRR) to distinguish healthy and stone-former urine samples. The methodof Zhmurov et al. has the obvious disadvantage of analyzing changes incomplex patterns of phospholipid composition. With regard to the Azouryet al. method, the high cost of nuclear magnetic resonanceinstrumentation is a major contributing factor to its generalunavailability in clinical laboratories.

The techniques described either by Zhmurov et al. or Azoury et al. havenot been instituted as a commercial diagnostic measure. Morefundamentally, the changes reported by Zhmurov et al. and Azoury et al.may relate to secondary pathophysiologic responses to the presence ofkidney stones or to the absence of dicitrate cyclic diester (seediscussion below), and they may not be generally applicable prior to theoccurrence of stone disease.

The type of calculus evidenced in any particular patient is dueprimarily to a urinary deficiency of inhibitors normally present to helpsuppress crystal formation. Several compounds have been proposed ascontributing factors in the inhibition of crystal formation. Forexample, Schreier et al. reported that about one-half of the inhibitoryactivity for calcium oxalate crystal growth in urine was due tocompounds smaller than 5000 daltons, and that a portion of the activityhad been shown to be due to pyrophosphate and citrate. Large molecularweight inhibitors in three families, e.g., glycosaminoglycans (GAGs),ribonucleic acids (RNAs), and glycoproteins, accounted for approximately40% of total inhibition. The remainder of inhibitory activity was due tocompounds which were chemically undefined. Thus, a variety of compoundshave been suggested as potential inhibitors of kidney stones (Schreier,E. E., K. E. Lee, J. L. Rubin, P. G. Werness, L. H. Smith [1979]"Macromolecular Inhibitors of Calcium Oxalate Crystal Growth andAggregation in Urine," In Oxalate in Human Biochemistry and ClinicalPathology (G. A. Rose et al, eds.), London, The Welcome Foundation, pp.22-610).

It has been known for decades that urine can prevent cement fromhardening properly. One of the current inventors described isolation,using column chromatography, of a fraction from urine which appeared toinhibit setting of cement. A citride compound was synthesized whichreduced the ability of cement to harden, but it was not determined whatthe exact structure of the compound was. The compound was determined tobe distinct from the water soluble di-, tri-, or tetramethyl citratecompounds isolated from pineapple in the course of preparing vitamin C,as described nearly sixty years ago. See Bose, P. K., S. N.Bhattacharyya (1936) Science and Culture 2:162. Moreover, it wasundetermined what the role of the compound was in the inhibition ofhardening of cement and whether there was any relation to kidney stoneformation (Thomas, Jr., W. C. [1988] Md. Med. J. 37:861-862). Nor was itknown whether this compound corresponded to a natural inhibitor orwhether the compound was a single chemical species. Furthermore, therewas no evidence then available to indicate that calcareous stone diseasecould be reliably linked to the absence of any single metabolite. Thespeculative nature of these studies proposing a role for inhibitors ofcalculus formation is evidenced by subsequent studies which continued tosuggest that proteins, especially glycoproteins, played an importantrole in the inhibition of calcium oxalate crystal formation (Coe, F. L.,Y. Nakagawa, J. H. Parks [1991] Am. J. Kidney Disease 17:407-413).However, epidemiological studies of certain substances, e.g., citrate,phosphate, pyrophosphate, glycosaminoglycans, nephrocalcin,Tamm-Horsfall proteins, and uropontin, showed that none of thesesubstances can be used as an unambiguous, single-determinant marker forcalcareous stone disease.

Accordingly, the development of prospective diagnostic techniques toidentify those patients who are predisposed to developing kidney stonesis needed to provide a means for mitigating these costs through theexercise of preventative practices by those predisposed patients.Therefore, the discovery of a singular, unambiguous marker as describedhereinbelow is a substantial contribution to the art.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to a method for distinguishing normal andkidney stone-forming patients based on the presence or absence of anovel compound, dicitrate cyclic diester or an analog, derivative,metabolic precursor, metabolite, or salt thereof. The presence of thiscompound in healthy patients and its absence in stone-forming patientsoffers definite advantages in its use as a diagnostic measure. Byanalyzing the dicitrate cyclic diester content in as little as 2-3 ml ofurine, a predisposition to stone formation can be diagnosed before theoccurrence of a stone episode. Kits which can be used, for example, fordiagnostic purposes, can comprise the substantially pure novel compoundor its analogs, derivatives, metabolic precursors, metabolites, orsalts, or other components used to detect the novel compound.

The subject compound can also be synthesized by a novel method whichemploys the reaction of citrate with a dehydrating agent, e.g.,dicyclohexyl carbodiimide.

Further, the subject compound, or a derivative, metabolite, or salt ofthe compound, and compositions comprising the subject compounds can beemployed in a novel method for inhibition of calcification applicable tothe prevention or treatment of calcification-related diseases, e.g.,kidney stone disease.

It is another object of the invention to utilize the metal ioncomplexing properties of the subject compounds to reduce theconcentration of free metal ions where they are undesired. Thus, it isconsidered that the subject invention includes the use of the subjectcompounds as complexing agents to bind with, and reduce the amount of,free metal ions associated with certain diseases or with metal toxicityor contamination. Complexing agents are also useful as inhibitors offood discoloration caused by the presence of metal ions. Therefore, thecompounds can also be used to complex metal ions in foods and preventthe discoloration of foods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an HPLC profile of synthetic dicitrate cyclic diester (8.3minutes) and citrate (6 minutes). The peak at approximately 4 minutes isthe injection, or solvent, peak.

FIG. 2a shows an HPLC elution profile of a urine sample obtained from ahealthy subject.

FIG. 2b shows an HPLC elution profile of a urine sample obtained from acalculous patient.

DETAILED DISCLOSURE OF THE INVENTION

We discovered a novel urinary metabolite in the urine of humans who showno symptoms of, or no predisposition to, renal calculous disease. Ourfindings demonstrated that the major urinary inhibitor of hydroxyapatitecrystal formation is a citrate-rich acidic compound having a molecularmass of less than 2,000 Daltons and having the general structure:##STR1## wherein R₁, R₂, R₃, and R₄ can be the same or different and canbe OH, O--(CH₂)_(a) CH₃, wherein n=1-50, O-aryl, NH, N-alkyl, or N-aryl;and X₁ and X₂ can be the same or different and can be O or NH. In apreferred embodiment, the compound is dicitrate cyclic diester, whereinR₁ =R₂ =R₃ =R₄ =OH and X₁ =X₂ =O. Dicitrate cyclic diester is alsoreferred to herein as "dicitrate" and means dicitrate cyclic diester andany analogous form, derivative, metabolite, or salt thereof. Theseanalogs, derivatives, metabolic precursors, metabolites, or salts of thesubject compound would be readily recognized by a person of ordinaryskill in the art. For example, a metabolic precursor would be recognizedas an uncyclized mono- or diester of the subject compound, or a proformof the subject compound which can include the compound or its precursorassociated with, or bound to, a peptide or coenzyme or other moleculewhich is cleaved from the compound when biologically transformed ormetabolized in the body. The compound formed by esterification ofcitrate with itself can also include uncyclized dimer, as well astrimers or other polymers. The novel compound can be isolated using anovel chromatographic procedure, as described herein, or can besynthesized by chemical or biotechnological procedures.

In chemical synthesis of the novel dicitrate cyclic diester compound,citrate and a dehydrating agent, e.g., dicyclohexyl carbodiimide,carbonyl diimidazole, other diimide-based agents and the like, can beused as starting materials. To synthesize the diester, the diimidecompound and citrate are typically provided at a ratio of 1:2. When thecitrate is provided at less than twice the molar amount of the diimide,e.g., at a ratio of 1:1 or less, a dicitrate monoester intermediate canbe formed. The monoester can be formed as an intermediate by metabolicprocesses in the body as well.

The novel compound can be synthesized by a dehydration reaction in thepresence of tetrahydrofuran (THF). Preferably, the citric acid and THFare provided in their anhydrous forms. Other compounds which can be usedin this reaction would be recognized by those of ordinary skill in theart. For example, other carboxylic acids, e.g., tartaric acid, isocitricacid, or 3-amino citric acid, can be used as starting material withtitrate to produce structural analogs of dicitrate. Other solvents,e.g., pyridine, and various aqueous buffers, can also be used in placeof, or in addition to, THF.

A novel method for detecting the presence of the subject compound in abiological sample is also described. Preferably, the biological sampleis subjected to a separation procedure as a clean-up step, essentiallyfractioning the dicitrate cyclic diester from undesired compounds in thebiological sample. The biological sample is typically urine or blood butcan be other body fluids, including saliva or other digestive fluids,lymphatic fluid, or other intra- or extracellular fluid or tissue wheredicitrate is present at detectable levels. The separation techniquecomprises the utilization of column chromatography which can furthercomprise a first step of separating the target compound from the sample,e.g., urine, on a gel permeation column. Typically, a chromatographycolumn using Sephadex G-25 as the stationary phase can be used to adsorbthe target compound in this initial step. The compound can be elutedfrom the stationary phase with a salt solution, preferably a carbonatesalt solution, for example, ammonium carbonate.

Citrate-containing fractions can then be further separated byion-exchange column chromatography. These citrate-containing fractionscan be determined colorimetrically. Due to the charge associated withcitrate, anion exchange resins are preferred as the stationary phase forthe ion-exchange chromatography step. The dicitrate fraction eluted fromthe ion-exchange column is then passed through a second gel permeationcolumn, eluted, and lyophilized. The lyophilized residue can then beredissolved in water for subsequent detection, which is preferably byhigh performance liquid chromatography (HPLC) analysis. A typical HPLCelution profile is presented in FIG. 1, wherein the three peaks abovezero absorbance are the solvent, or injection, peak (elution time 2-4minutes), the citrate peak (maximal at 6 minutes), and the dicitratecyclic diester peak (present at 8.3-8.6 minutes).

By applying these separation and detection methods to urine samples fromhealthy and calculous patients, we have observed that the dicitratecyclic diester peak is absent in samples from patients with renalcalculi (see FIGS. 2a-2b). Determinations of the presence of dicitratecyclic diester in urine were conducted in a completely blindedexperimental procedure and, in all cases, healthy patients showed clearevidence of a dicitrate cyclic diester peak, and kidney stone patientsshowed no dicitrate cyclic diester peak above the baseline of theelution profile. Furthermore, in one case where additional citrate wasadministered orally to a kidney stone patient, we observed a prominentcitrate peak, but no dicitrate cyclic diester was evident even thoughthe dietary intake of citrate was relatively high.

Thus, the presence or absence of dicitrate cyclic diester is anadvantageous and valuable diagnostic determinant for identifyingindividuals likely to develop kidney stones. Other disease conditionsincluding, but not limited to, formation of atheromas inarteriosclerosis, hypercoagulation of blood which has been indicated inmicrovasuclar stroke, calcium deposition on bone, e.g., bone spurformation, dental calculus formation, as well as the genesis ofgallstones, can also involve pathologic calcification processes, and areconsidered to be pathologic calcification conditions for purposes of thesubject invention. Dicitrate cyclic diester can be used as a clinicalmarker and for treatment of these conditions. Preferably, the presenceof dicitrate cyclic diester can be detected by the novel HPLC methoddescribed herein. However, the detection of dicitrate cyclic diester inhuman fluids can include the utilization of other methods including, butnot limited to, adsorption/ion-exchange chromatography, colorimetry,electrophoresis, ultraviolet/visible/infrared/Raman spectroscopy, gaschromatography, mass spectrometry, and immunochemical and biochemicaltechniques. These detection techniques can involve the production ofderivatives of dicitrate prior to analysis.

Kits comprising a separately packaged dicitrate, precursor, metabolite,or reactant can be advantageously used in the diagnosis and treatment ofpathologic calcification conditions. For example, a simple colorimetrictest for diagnosing the presence of dicitrate can be packaged as a kitwhich comprises neutral 4M hydroxylamine and ferric chloride solution.Following the mixture of the biological sample to be tested with thehydroxylamine, ferric chloride solution can be added to yield a coloredreaction product detectable at approximately 540 nm. The detection ofdicitrate cyclic diester can result from direct or indirect detection ofthe compound. Therefore, it should be understood that other forms of thecompound can be what is actually detected. For example, detection of ametabolic precursor or intermediate of dicitrate cyclic diester or ametabolic product therefrom can be the equivalent of detecting thecompound itself. Moreover, the detection of an enzyme which can catalyzethe biosynthesis of dicitrate or, alternatively, detecting the absenceof an enzyme which can metabolize dicitrate to its breakdown product(s)can also effectively be used to detect the presence or absence ofdicitrate.

It is also understood that antibodies can be made to the novel dicitratecompound, or its analog, derivative, or salt, or antibodies to ametabolic enzyme involved in the biosynthesis or biotransformation ofdicitrate. These procedures for antibody production are standard in theart and could be readily conducted by persons of ordinary skill in theart. For example, antibodies can be raised to the dicitrate compounditself or using the compound as a hapten conjugated to an acceptablecarrier, wherein the carrier is an immunogen. These antibodies can beuseful for purification procedures, e.g., affinity chromatography, forimmunoassay or diagnostic kits, for therapeutics, and other methods andprocedures where antibodies typically can be employed. ,An assayemploying specific antibodies to detect dicitrate can involverecognition, i.e., binding, of the antibody to the compound or thecompound-specific moiety of a hapten-carrier conjugate. The antibody canbe labelled by any available means, e.g., radioactive or enzymaticmarkers, or can be further recognized by a second antibody having alabel.

Dicitrate cyclic diester is a potent inhibitor of hydroxyapatite andcalcium-oxalate crystal formation. Our studies indicate that at leastone step in stone formation (i.e., nucleation, growth, aggregation, orcrystal retention on renal tubular surfaces) is blocked by this novelrenal calcification inhibitor (see Example 5). Novel treatments forkidney stone disease and other pathologic calcification processes canalso be accomplished by administration of dicitrate cyclic diester,analogs, metabolites, derivatives, or salts thereof, or compositionscomprising the compound or one of its analogs, derivatives, metabolites,or salts. Thus, dicitrate can be useful for prophylactic or therapeutictreatment of a calcification process, including idiopathic kidney stonedisease or other pathologic calcification.

The treatment of patients with pathologic calcification diseases, orsymptoms thereof, can be carried out by administering to these patientsan effective amount of the compound, itself, or its metabolic precursor,analog, derivative, metabolite, or salt, or a composition comprising thenovel compound, its precursor, analog, derivative, metabolite, or salt.The compound used in these compositions can be isolated and purifiedfrom biological sources, can be chemically synthesized, or can beproduced using biotechnology procedures.

Preferably, the novel compound is therapeutically administered orally orparenterally or can be provided as a dietary supplement. However, asexemplified below, other routes of administration of the novel compoundcan also be employed.

Because chelation of a metal ion, e.g., Fe(II), Fe(III), Al(III), andthe like, results in high affinity complexes, dicitrate cyclic diester,which exhibits chelating activity can be useful as a metal ioncomplexing agent in controlling, i.e., lowering, the concentration ofthese and other free metal ions which are associated with pathologicalconditions. Pathological conditions associated with abnormal free metalion concentrations include, for example, Wilson's disease, Alzheimer'sdisease, and reperfusion injury often seen in patients with heart attackor spiral injuries.

The methodology for complexing metal ions can also be used in treatingheavy metal poisoning in an animal or human such as that seen withcadmium, lead, strontium, arsenic, and the like. It would also beunderstood that the subject compounds can be used as a complexing agentto bind with, and thus reduce the concentration of free metal ions,including heavy metal contaminants in the environment. For example, thedicitrate can be used as a treatment or in a filter to remove heavymetals from an environmental substance, such as water, soil, or airwhere heavy metals may be present, by contacting the compounds with thebiological or environmental substance.

It is also known that certain free metal ions can discolor food. Thus,dicitrate can also be used as a complexing agent to reduce the freemetal ion concentration in foods, thus inhibiting the process resultingin food discoloration. The methodology employed in these additional usesfor dicitrate as a free metal ion complexing agent would be readilyunderstood by those of ordinary skill in the art.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Isolation of Dicitrate Cyclic Diester from Urine or Serum Samples

An aliquot (20 ml) of urine collected over the 24-hour period (eachspecimen combined and refrigerated during the collection) was passedthrough a Sephadex G-25 gel permeation column. The sample was elutedwith 5 mM ammonium carbonate, and the citrate-containing fractions wereidentified colorimetrically, then pooled, and lyophilized. The samplewas redissolved in water and passed through a Dowex AG-1X8anion-exchange chromatography column. Samples were eluted withincreasing concentrations of ammonium carbonate (linear gradient from100 mM to 700 mM), and the citrate-containing fractions were againpooled and lyophilized. The solid is redissolved in water and passedthrough a second Sephadex G-25 column, and citrate-containing fractionswere pooled and lyophilized. An identical procedure is used to obtainthe major calcification inhibitor fraction from a 20 ml sample of humanserum.

EXAMPLE 2

High Performance Liquid Chromatography Analysis

The citrate-containing fractions obtained by the column chromatographicmethods described above were redissolved in 0.5-1.0 ml water and appliedto a C-18 reverse-phase chromatography column using a Hewlett-Packard1090a high volume liquid chromatograph. Samples were elutedisoelectrically with one percent (volume/volume) trifluoroacetic acid inacetonitrile, and detected by UV absorbance at 214 nm.

EXAMPLE 3

Detection of Dicitrate Cyclic Diester in Biological Samples

Following separation procedures as described herein, HPLC analysis ofpatients' urine samples consistently showed the complete absence of thedicitrate cyclic diester peak in those patients with renal calculi.Illustrative are the results of one set of determinations with a healthysubject and a calculous patient shown in FIGS. 2a and 2b. We conductedadditional blind studies, of which the experimental results weresubsequently decoded by one or more of the inventors. In all cases,healthy patients showed clear evidence of a dicitrate cyclic diesterpeak, whereas kidney stone patients had no discernable dicitrate cyclicdiester peak above the baseline of the elution profile. Even stonepatients having relatively high citrate supplements added to their dietdid not subsequently show a dicitrate cyclic diester peak on HPLCanalysis. Therefore, the absence of dicitrate cyclic diester in a stonepatient does not result simply from reduced availability of a citrateprecursor. Moreover, two patients with uric acid stones (a conditionwhich is not at all related to calcareous renal calculi) were observedto have dicitrate cyclic diester in their urine. We also found thatpatients who form calcium oxalate stones as a result ofhyperparathyroidism have dicitrate cyclic diester in their urine. Thus,dicitrate cyclic diester determinations can be used as a marker ofidiopathic calcium oxalate urolithiasis, and allow the idiopathic stoneformation to be distinguished from other hormone-related kidney stonedisease. The results of these studies are shown in Table 1, below.

                  TABLE 1                                                         ______________________________________                                        A summary of clinical dicitrate determinations by HPLC                                                         Detectable                                   Subject                                                                             Age    Sex    Diagnosis    Urinary DCD                                  ______________________________________                                        D. C. 32     M      Idiopathic Stones                                                                          No                                           N. C. 32     M      Idiopathic Stones                                                                          No                                           L. Q. 37     F      Idiopathic Stones                                                                          No                                           R. N. 33     M      Idiopathic Stones                                                                          No                                           J. A. 37     M      Incomplete RTA                                                                             No                                           B. P. 32     M      Normal       Yes                                          L. H. 31     M      Normal       Yes                                          W. T. 63     M      Normal       Yes                                          L. S. 50     F      Normal       Yes                                          B. B. 39     M      Normal       Yes                                          B. G. 39     F      Uric Acid Stones                                                                           Yes                                          B. S. 57     M      Uric Acid Stones                                                                           Yes                                          V. C. 59     M      Hyperparathyroidism                                                                        Yes                                          R. K. 50     F      Hyperparathyroidism                                                                        Yes                                          ______________________________________                                         DCD = dicitrate cyclic diester                                           

All of our experimental results consistently segregate healthy andstone-forming subjects, and they demonstrate the utility of our methodin the prospective detection of kidney stone patients.

EXAMPLE 4

Application of Dicitrate to Other Pathological Calcification Processes

The dicitrate compound, and methods of use involving detection of thecompound or its prophylactic or therapeutic administration, have beendescribed herein as primarily relating to renal calculus. Otherimportant pathologic calcification processes can also benefit from thedetection or administration of dicitrate. These include, but are notlimited to, the following conditions:

A. Coagulation.

Blood coagulation is exquisitely sensitive to the concentration ofcalcium ion which is an essential activator of many calcium-activatedenzymes responsible for both intrinsic and extrinsic clotting pathways.A steady normo-calcemic state in humans is effectively maintained by twohormonal factors: (a) calcitonin, which inhibits bone calciummobilization, thereby suppressing a rise in circulating calcium ionconcentration; and (b) parathyroid hormone, which mobilizesbone-dissolving osteoclasts to release calcium into circulation andwhich enhances renal tubular reabsorption of calcium ion. Because ioniccalcium (i.e., uncomplexed Ca²⁺) is the most likely feedback signal,circulating calcium ion-binding metabolites can act as hypocalcemicagents and suppress coagulation. The hitherto unrecognized absence ofdicitrate cyclic diester in calcareous stone patients promoteshypercoagulation. Thus, elevation of dicitrate concentration in theblood of stone patients comparable to normal circulating concentrationsof dicitrate in the blood of healthy subjects can be useful insuppressing coagulation. Inhibition of hypercoagulation is a recognizedtreatment or preventive measure against microvascular stroke. Dicitratecan therefore be an especially important substitute for heparin sulfate,which is typically used clinically to inhibit coagulation duringangioplasty and other procedures requiring suppression of patient bloodclotting.

B. Arteriosclerosis.

Hardening of the arteries (or arteriosclerosis) is acirculation-obstructing disease involving calcification. One example isintimal arteriosclerosis in which fatty deposits form in the bloodvessel lining followed by pathologic calcification of the arterialmiddle layer (or media). Another form of arteriosclerosis occurs as aresult of scar tissue formation on the vessel lining as well ascalcification. Ultimately, loss of vessel elasticity and concomitantincrease in blood pressure arises as the level of calcification hardensthe blood vessel lining. Because dicitrate cyclic diester suppressespathological calcification, our discovery that dicitrate is present inhealthy patient blood indicates that this agent can be used to suppressarteriosclerosis in healthy patients. Therefore, elevation ofcirculating dicitrate to concentrations can inhibit the course andseverity of arterial vessel hardening.

C. Dentifrices/Calculus-Inhibiting Mouthwashes.

Because oral pathological calcification is a causative factor inperiodontal disease, various toothpastes and mouthwashes are formulatedwith pyrophosphate salts which bind free calcium and thereby reduce thethermodynamic driving force for calculus formation on teeth. Ourdiscovery of dicitrate cyclic diester as a hitherto unrecognizednaturally occurring inhibitor of pathological calcification indicatesthat dentifrice and mouthwash formations can be a beneficial treatmentin the prevention of calculus accumulation. Furthermore, becausedicitrate acts in two different ways (i.e., binding to free calcium ionand binding to calcified surfaces), this metabolite can offeradvantageous properties as compared with exclusively calcium ion-bindingagents such as pyrophosphate.

EXAMPLE 5

Non-Therapeutic Application of Dicitrate

In view of the advantageous calcium-binding properties of dicitrate, itwould be recognized by those of ordinary skill in arts related to, aswell as unrelated to, the medical arts that dicitrate, or compositionscomprising dicitrate, can have utility in non-therapeutic applications.For example, in the arts of laboratory science or blood productanalysis, collection, or utilization, the dicitrate can be used as ananticoagulant in blood collection tubes, in intravenous orintra-arterial catheters, or other like applications where coagulationof blood can be detrimental to the work being conducted.

Alternatively, inhibition of calcification processes can be useful innon-medical fields. For example, calcification on the interior walls ofwater pipes or plumbing facilities can create problems, especially ingeographic areas where high calcium is present in the water supply.Dicitrate has the advantageous properties of binding free calcium ionsand thereby can prevent the buildup of calcium on the inner walls ofwater pipes and the like. A composition comprising dicitrate can beapplied to water lines or can be coated onto or incorporated into thewater pipes or plumbing fixtures to resist the initiation of thecalcification process. Thus, it is contemplated by the inventors thatthe dicitrate and compositions comprising dicitrate can be used in waysto prevent these non-pathologic calcification processes. Othercalcification processes which involve free calcium ion or calcium saltdeposition can also be inhibited by the subject dicitrate.

EXAMPLE 6

Synthesis of Dicitrate Cyclic Diester

Anhydrous citric acid (5 mmoles, 960 mg) and dicyclohexyl carbodiimide(10 mmoles, 2.06 g) were stirred in 30 ml anhydrous tetrahydrofuran forone hour. Dicyclohexylurea crystals formed during the ensuingdehydration reaction were removed by filtration, and the filtrate wasdried in vacuo, yielding the dicitrate cyclic diester-containing solidresidue.

The synthetic conversion of citric acid and dicyclohexyl carbodiimideinto dicitrate cyclic diester was confirmed both by HPLC elutioncharacteristics and as mass spectrometry. As shown in FIG. 1, dicitrateand citrate have well-defined chromatographic properties, and theadditional ionic charge of dicitrate results in its correspondinglygreater retention time for isocratic elution. In addition, the mass ofthe molecular ion was found to be 348.1 using a Finnigan time-of-flightquadrapole mass spectrometer, equipped for laser desorption of ionicsamples. This value corresponds to mass predicted on the basis of thedicitrate cyclic diester structure.

EXAMPLE 7

Dicitrate Inhibition of Calcification

Dicitrate demonstrates potent inhibitory properties in two differentcalcification assays. In the first, we used a tendon mineralizationassay with supersaturated solutions of calcium and phosphate (Thomas, W.C., Jr., A. Tomita [1967] Am. J. Pathol. 51:621,628). Bovine Achillestendon served as the biological matrix material for mineralization inbuffered solutions containing calcium ion (3-7 mg/liter) and phosphate(2-5 mg/liter) in the absence or presence of 3-5 mM citrate ordicitrate. In this assay, dicitrate was 12-14 times more effective thanan equivalent concentration of citrate in inhibiting the formation ofcrystals in the collagen fibrils of the tendon.

In the second calcification protocol (Antinozzi, P. A., C. M. Brown, D.L. Purich [1992] J. Crystal Growth 125:215-222), calcium chloridedihydrate and potassium oxalate are added to a buffered solutionconsisting of 0.1M sodium chloride and 0.01MN,N'-hydroxyethylpeperazine-ethanesulfonate (pH 6.5) at a relativesupersaturation (i.e., the calcium oxalate ion-activity product dividedby the equilibrium solubility product) of 18. The presence of 5 mMdicitrate inhibited the rate of calcium oxalate crystallization by afactor of 5-8 relative to an equivalent concentration of citric acid.

EXAMPLE 8

Formulations

The compounds of the invention are useful for various non-therapeuticand therapeutic purposes. It is apparent that the compounds of theinvention are effective for inhibiting kidney stone formation and forother calcification process-related diseases. Specifically exemplifiedherein is the control of idiopathic renal calculous disease.

Therapeutic application of the new compounds and compositions containingthem can be contemplated to be accomplished by any suitable therapeuticmethod and technique presently or prospectively known to those skilledin the art. Further, the compounds of the invention have use as startingmaterials or intermediates for the preparation of other useful compoundsand compositions.

The dosage administration to a host in the above indications will bedependent upon the identity of the calcification disease and the age,health, and weight of the patient as well as the kind of concurrenttreatment, if any, frequency of treatment, therapeutic ratio, and likeconsiderations.

The compounds of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive compound(s) is combinedwith a suitable carrier in order to facilitate effective administrationof the composition.

The compositions of the invention are advantageously used in a varietyof forms, e.g., tablets, ointments, capsules, pills, powders, aerosols,granules, and oral solutions or suspensions and the like containing theindicated suitable quantities of the active ingredient. Suchcompositions are referred to herein and in the accompanying claimsgenetically as "pharmaceutical compositions." Typically, they can be inunit dosage form, namely, in physically discrete units suitable asunitary dosages for human or animal subjects, each unit containing apredetermined quantity of active ingredient calculated to produce thedesired therapeutic or prophylactic effect in association with one ormore pharmaceutically acceptable other ingredients, e.g., diluent orcarrier.

Where the pharmaceutical compositions are aerosols, the activeingredients can be packaged in pressurized aerosol containers with apropellant, e.g., carbon dioxide, nitrogen, propane, etc. with the usualadjuvants such as cosolvents, wetting agents, etc.

Where the pharmaceutical compositions are ointments, the activeingredient can be mixed with a diluent vehicle such as cocoa butter,viscous polyethylene glycols, hydrogenated oils, and such mixtures canbe emulsified if desired.

In accordance with the invention, pharmaceutical compositions comprise,as an active ingredient, an effective amount of one or more non-toxic,pharmaceutically acceptable ingredient(s) or carriers.

Examples of such carriers for use in the invention include ethanol,dimethyl sulfoxide, glycerol, silica, alumina, starch, and equivalentcarrier and diluents. While effective amounts may vary, as conditions inwhich such compositions are used vary, a minimal dosage required foractivity is about 50 micrograms. To provide for the administration ofsuch dosages for the desired therapeutic treatment, new pharmaceuticalcompositions of the invention will advantageously comprise between about0.01% and about 50%, and especially, about 0.1% and about 30%, by weightof the total of one or more of the new compounds based on the weight ofthe total composition including carrier or diluent. Illustratively,dosage levels of the administered active ingredients can be:intravenous, 0.01 to about 20 mg/kg; intraperitoneal, 0.01 to about 100mg/kg; subcutaneous, 0.01 to about 100 mg/kg; intramuscular, 0.01 toabout 100 mg/kg; orally 0.01 to about 200 mg/kg; dermally 0.01 to about500 mg/kg and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

The compounds of the subject invention can be parenterally, orally, ortopically administered to subjects requiting treatment. The activecompounds may be mixed with physiologically acceptable fluids such assaline or balanced salt solutions. Also, solid formulations such astablets or capsules can be made using enteric coatings or otherexcipients which are known in the art.

The compounds of the subject invention may be applied, for example,orally, intravenously, intramuscularly, intraperitoneally, intranasally,intradermally, or subcutaneously. The compounds of the subject inventionmay also be combined with other substances to provide enhancedtreatment.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. A dentifrice for inhibiting dental plaque formation ordental calculus, said dentifrice comprising dicitrate cyclic diesterhaving the general structure ##STR2## wherein R₁ =R₂ =R₃ =R₄ =OH and X₁=X₂ =O, or a salt thereof.